Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: HEK293 cells were transfected with a vector expressing human TfR1 (alongside EGFP). Subsequently, cells were treated with each TfR1b-Nb at a concentration of 400 nanomolar, followed by incubation with an anti-His-APC antibody. The two nanobodies highlighted in grey are the parental sequences from which the final selected NewroBus molecules were derived.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Transfection, Plasmid Preparation, Expressing, Concentration Assay, Incubation, Derivative Assay

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: The upper-left panel demonstrates the dose-dependent competition of unlabeled Transferrin for binding to TfR1 on the cell surface of CHEK-ATP089 cells in the presence of FITC-Transferrin. Subsequent panels assess the inhibitory activity of 14 TfR1b-Nbs from Families A, B, D, G, and I. A nanobody from Family L (which lacks binding to TfR1 on CHEK-ATP089 cells) serves as a negative control. In these experiments, CHEK-ATP089 cells were incubated with the specified proteins for 1 hour on ice before FACS analysis. Human Transferrin-FITC and TfR1b-Nbs were utilized at a concentration of 2.5 micromolar.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Binding Assay, Activity Assay, Negative Control, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: The panel demonstrates the dose-dependent competition of unlabeled Transferrin pHrodo Red-TF uptake by CHEK-ATP089 cells. The right panel assess the inhibitory activity of representative TfR1b-Nbs from Families A, B, D, G, and I. A nanobody from Family J (which lacks binding to TfR1 on CHEK-ATP089 cells) serves as a negative control.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Activity Assay, Binding Assay, Negative Control

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: A) ELISA detection of TfR1b-Nbs in brain homogenates of rats expressing human TfR1 ( Tfr1 h/w or Tfr1 h/h ), but not in wild-type rats ( Tfr1 w/w ), 16-18 hrs. following IV injection. B) Detection of TfR1b-Nbs 04B05R3 (Family A) and 05D01R3 (Family D) in serum, CSF, and brain homogenates of Tfr1 h/w and Tfr1 w/w rats 44–48 hours post-injection. Both nanobodies showed high CSF/serum ratios in Tfr1 h/w rats, indicating efficient BBB penetration and sustained stability.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, IV Injection, Injection

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet:

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Expressing

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: A) ELISA quantification of TNFI-Nb1 in serum and brain homogenates from Tfr1 w/w and Tfr1 h/w rats 6 hours after intravenous injection. TNFI-Nb1 is detected in serum but not in brain tissue, indicating that it does not cross the BBB. B) SDS-PAGE and Coomassie Blue staining of equal volumes of serum and CSF from Tfr1 h/h rats. While serum contains abundant proteins, only a faint ∼70 kDa band is detected in the CSF, confirming the low protein content of CSF and intact BBB exclusion of serum proteins.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, SDS Page, Staining

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: A) Schematic of the heterodimer composed of TNFI-Nb1 fused to TfR1b-Nb 04B05R3 (Family A), produced in CHO-S cells. B) ELISA-based detection of the heterodimer in serum, CSF, and brain homogenates of Tfr1 h/w rats, but not in Tfr1 w/w controls, following IV injection. The heterodimer exhibited efficient BBB penetration with a CSF/serum ratio of ∼1, confirming human TfR1-dependent CNS delivery. Samples were analyzed 48 hours post-IV injection.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Produced, Enzyme-linked Immunosorbent Assay, IV Injection

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: A) Western blot validation of brain fractionation into vasculature and parenchyma. Glut1 (endothelial marker) was enriched in the vasculature fraction, while Gapdh and cell-type–specific markers—Vamp2 and NmdaR2b (neurons), Iba1 (microglia), Eaat2 (astrocytes), and Mbp (oligodendrocytes)—were enriched in the parenchymal fraction. Human TfR1 was detected in all fractions, with strongest signal in the vasculature. B) ELISA quantification of TNFI-Nb1–04B05R3 in homogenate, vasculature, and parenchymal fractions. After normalization to protein content, the parenchymal fraction showed the highest nanobody levels, supporting uptake by CNS cells via human TfR1-mediated transcytosis.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Western Blot, Fractionation, Marker, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: A ) Anti-His tag antibody (green) and ( B ) anti-human TfR1 antibody (red) stain TNFI-Nb1-linker-04B05R3 and human TfR1, respectively, in the brains of Tfr1 h/w but not Tfr1 w/w rats. C ) Human TfR1 (red), CD31 (purple)—an endothelial cell marker—and TNFI-Nb1-linker-04B05R3 (green) colocalize in vessels in the brains of Tfr1 h/w rats. White arrows in the zoomed-in panel indicate the colocalization spots within the region highlighted by the white square in the overlay panel.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: Sections were stained with an anti-His tag antibody (green) to detect TNFI-Nb1-linker-04B05R3 and an anti-GFAP antibody (red) to label astrocytes. TNFI-Nb1-linker-04B05R3 is present in the brains of Tfr1 h/w rats but not in Tfr1 w/w rats. In Tfr1 h/w rats, TNFI-Nb1-linker-04B05R3 colocalizes with some astrocytes, indicated by yellow overlays in the merged images. White arrows in the zoomed-in panel point to colocalization spots within the region highlighted by the white square in the overlay panel.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Staining

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: Sections were stained with an anti-His tag antibody (green) to detect TNFI-Nb1-linker-04B05R3 and an anti-IBA1 antibody (red) to label microglia. TNFI-Nb1-linker-04B05R3 is present in the brains of Tfr1 h/w rats but not in Tfr1 w/w rats. In Tfr1 h/w rats, TNFI-Nb1-linker-04B05R3 colocalizes with most microglia, as indicated by yellow overlays in the merged images. White arrows in the zoomed-in panel point to colocalization spots within the region highlighted by the white square in the overlay panel.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Staining

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: HEK293 cells were transfected with a vector expressing human TfR1 alongside EGFP. The cells were then treated with each TfR1b-Nb at a concentration of 400 nM, followed by incubation with an anti-His-APC antibody. Secondary antibody staining alone is shown as a negative control. Binding of the wild-type TfR1-Nb from which the mutants originated is also shown for comparison.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Transfection, Plasmid Preparation, Expressing, Concentration Assay, Incubation, Staining, Negative Control, Binding Assay, Comparison

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: All eight TfR1b-Nbs exhibited elevated CSF levels following IV injection, confirming human TfR1-dependent BBB transcytosis. Among them, 05F02_enhanced WT-CDR3STEM-W showed the highest CSF/serum ratio. Details of the animals and nanobody administration are provided in .

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: IV Injection

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: A ) Schematic representation of the 16 humanized and optimized TNFI–TfR1b heterodimers. B ) Flow cytometry analysis in HEK293 cells stably expressing human TfR1, comparing the binding of heterodimers to that of their parental humanized TfR1b-Nbs. C ) Dose-response apoptosis inhibition curves in WEHI-13VAR cells treated with human TNFα and 2-fold serial dilutions of TNFI–TfR1b heterodimers (100,000 to 12.21 pM). Apoptosis was measured using a fluorogenic caspase-3/7 activation assay. The IC₅₀ values of the heterodimers were comparable to those of the parental humanized nanobodies TNFI-α (IC₅₀ = 48.84 pM, range: 42.11–56.56) and TNFI-β (IC₅₀ = 64.31 pM, range: 57.17–72.31).

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Flow Cytometry, Stable Transfection, Expressing, Binding Assay, Inhibition, Activation Assay

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: Tfr1 h/w and Tfr1 w/w rats were injected subcutaneously with TNFI-β–TfR1b-A2 ( A ) and TNFI-β– TfR1b-D1 ( B ). Heterodimers levels were measured 72 hours post-injection. TNFI-β–TfR1b-A2 achieved higher serum and CSF levels (CSF/serum ratio: 0.14), while TNFI-β–TfR1b-D1 showed superior BBB permeability (CSF/serum ratio: 0.37). Heterodimers remained detectable in both CSF and serum three days post-injection, confirming prolonged in vivo stability. Tissue distribution was primarily brain-specific and human TfR1-dependent, with additional enrichment observed in the heart and, potentially, the kidney. with some enrichment in the heart and, potentially, the kidney.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: Injection, Permeability, In Vivo

Journal: bioRxiv

Article Title: NewroBus for the brain: humanized TfR1-targeting nanobodies with high BBB permeability and cargo transport capacity

doi: 10.1101/2025.04.20.649139

Figure Lengend Snippet: CBC analysis on ∼4-month-old rats humanized for both TF and TfR1 ( Tf h/w :Tfr1 h/h ), following administration of TNFI-β–TfR1b-A2 or PBS. No significant changes were observed in key hematological parameters—including RBC, hemoglobin (HGB), and hematocrit (HCT)—compared to PBS-treated controls. All values remained within normal physiological ranges, indicating no detectable hematotoxicity. These results suggest that TNFI-β–TfR1b-A2 is well tolerated in vivo and unlikely to induce anemia through interference with human TF–TfR1–mediated iron transport.

Article Snippet: Membranes were blocked with 5% non-fat milk (Bio-Rad, 1706404) in PBST (PBS + 0.05% Tween-20) for 45 minutes, then incubated overnight at 4°C with primary antibodies (1:1000 dilution in blocking buffer): Anti-human TfR1 (CST, 13113) Anti-Glut1 (CST, 73015) – endothelial cell marker Anti-GAPDH (Sigma, G9545) – loading control Anti-VAMP2, NMDAR2B (CST, 4212) – neuronal markers Anti-IBA1 (Wako, 01620001) – microglial marker Anti-EAAT2 (Synaptic Systems, 250203 or 104202) – astrocytic marker Anti-MBP (CST, 78896) – oligodendrocyte marker After washing, membranes were incubated for 45 minutes at room temperature with HRP-conjugated secondary antibodies (anti-rabbit: CST 7074 or Southern Biotech OB405005, 1:1000 in 5% milk).

Techniques: In Vivo